Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
A three-generation reproduction study of Ponceau 4R in the rat
Author:
Brantom P.G. et al.
Year:
1987
Bibliographic source:
Fd Chem. Toxic. 25(12, pp. 963-968

Materials and methods

Principles of method if other than guideline:
3-generation reproduction study in rats by oral route through diet. In each generation, ca. 1/3 of the females from each group were killed before parturition to provide data of in utero development. Foetuses of these animals were examined for skeletal abnormalities.
Remaining animals were allowed to litter and offspring were monitored for 21 days after birth for survival and development.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Housing: polypropylene and stainless-steel grid-floored cages, except during mating and rearing when the F0 and F1 generations were housed in aluminium cages with galvanized
grid-floors while the F2 generation used solid-floored polypropylene and stainless-steel cages. All gridfloored cages were suspended in racks over trays lined with paper on which excreta were collected, this paper was changed daily. Sawdust bedding used in the solid-floored cages was replaced as necessary. Shredded paper was provided as nesting material for pregnant females.
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C
- Air changes: 15-20 per hr; mating of F2 animals and rearing of F2 animals and rearing of F3 generation were carried out in rooms with 6-8 air changes/hr.
- Photoperiod (light/dark): 14/10 hrs

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: mixed with basal diet

DIET PREPARATION
- Dietary concentrations: adjusted weekly to maintain appropriate intakes; a sample of each diet was analysed prior to use

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 10-12 days
- After successful mating each pregnant female was caged: individually
- Other: for teratology phase, females were smeared each day until the day of mating was established
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sample of each prepared diet was analysed prior to use.
Duration of treatment / exposure:
- maintenance phase
F0: 64 days
F1: 56 days
F2: 57 days

- mating phase: 10-12 days, either random mating or time-mating (for teratology study)
Frequency of treatment:
Daily
Details on study schedule:
- F1a parental animals were maintained not mated until 56 days after selected from the F1 litters.
- Selection of parents from F1 generation: random
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Treated group: 36 rat/sex
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: yes, for any abnormality of appearance or behaviour
- Time schedule: daily

BODY WEIGHT: yes
- Time schedule for examinations: weekly (maintenance phase)

WATER AND FOOD INTAKE: yes
- Time schedule: twice-weekly (maintenance phase)

FOOD CONSUMPTION AND COMPOUND INTAKE:
Where rats of more than one age or sex were caged together diets were provided to give the appropriate substance intake to the smallest occupant, but it was assumed that rats less than 14 days old did not take diet directly. Dietary concentrations were adjusted weekly if necessary to maintain the appropriate mg/kg/day intakes.
Postmortem examinations (parental animals):
SACRIFICE OF:
- F0 adults upon remate: time-mated males part of teratology study and parents of F1b; time-mated females killed on day 20
- F1 adults; time-mated females killed on day 20
- F2 adults; time-mated females killed on day 20

All sacrificed rats were exposed to full autopsy

GROSS NECROPSY
Time-mated females.

FULL EXAMINATION
Animals to be subjected to a full post-mortem examination were weighed after fasting overnight (with free access to water).
The adrenal glands, brain, caecum with and without contents, gonads, heart, kidneys, liver, pituitary, spleen and stomach were weighed. The thyroid gland was weighed. After being weighed, the organs were preserved in 10 % neutral buffered formalin together with samples of aorta, colon, eye, Harderian gland, lungs, lymph nodes (cervical and mesenteric), mammary tissue, oesophagus, pancreas, rectum, salivary gland, skeletal muscle, skin, small intestine, spinal cord, thymus, trachea, urinary bladder, vena cava and seminal vesicles and epididymis or uterus and vagina. Any other tissue that appeared to be abnormal was also preserved. Abnormalities in the macroscopic appearance of the tissues were recorded. If the clinical condition or behaviour of a rat appeared abnormal prior to the post-mortem examination this also was recorded.

Postmortem examinations (offspring):
SACRIFICE OF:
- F1a young rats not selected as parental animals were subjected to gross autopsy
- F1b young rats: 1/sex/litter were killed on day 25 ± 1 and subjected to full autopsy; remainder rats were subjected to gross autopsy
- F2 young rats: 1/sex/litter were killed on day 27 ± 1 and subjected to full autopsy; remainder rats were subjected to gross autopsy
- F3 young adult rats: 2/sex/litter were killed on day 73 ± 2 and subjected to full autopsy; remainder rats were subjected to gross autopsy

GROSS NECROPSY
Animals subjected to a gross post-mortem examination were allowed food and water ad libitum up to the time of death. A macroscopic examination was made of all tissues listed below for the full examination except the brain, pituitary and spinal cord. Abnormal tissues were preserved in 10 % neutral buffered formalin.

FULL EXAMINATION
Animals to be subjected to a full post-mortem examination were weighed after fasting overnight (with free access to water).
The adrenal glands, brain, caecum with and without contents, gonads, heart, kidneys, liver, pituitary, spleen and stomach were weighed. The thyroid gland was weighed in the adult rats but not in the F1b and F2 generation pups. After being weighed, the organs were preserved in 10 % neutral buffered formalin together with samples of aorta, colon, eye, Harderian gland, lungs, lymph nodes (cervical and mesenteric), mammary tissue, oesophagus, pancreas, rectum, salivary gland, skeletal muscle, skin, small intestine, spinal cord, thymus, trachea, urinary bladder, vena cava and seminal vesicles and epididymis or uterus and vagina. Any other tissue that appeared to be abnormal was also preserved. Abnormalities in the macroscopic appearance of the tissues were recorded. If the clinical condition or behaviour of a rat appeared abnormal prior to the post-mortem examination this also was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues collected from the full autopsy of F3 control and high-dose animals were processed for wax embedding and sections were stained with haematoxylin and eosin for examination by light microscopy.
Statistics:
All results collected during this study were analysed using appropriate statistical techniques and a probability level of 0.05 or less was taken as statistically significant.
Reproductive indices:
In teratology study, it was recorded: number of corpora lutea in each ovary, number and position of foetuses and resorptions in each uterine horn, foetal viability, and the presence of any external foetal malformations.
Each resorption was classified as early or late, depending on the presence or absence of visible foetal tissue. Each viable foetus was weighed and preserved in 95 % alcohol. After fixation, foetuses were eviscerated and the eyes, the skin and supra-scapular fat were removed. Sex was determined during evisceration by examination of the gonads.
Ossified parts of the eviscerated foetuses were stained with Alizarin Red S using an automatic tissue processor. Foetuses were then examined using a dissecting microscope, and skeletal abnormalities and variations in the degree of ossification were recorded.
Offspring viability indices:
Number of pups in each litter was recorded on the day of birth and at 7, 14 and 21 days after birth. Litters, apart from those of F2 parents, were weighed and sexed on days 7, 14 and 21 after birth. Pup development was monitored by the use of developmental milestones, i.e. eyes open, ears unfurled, teeth erupted and hair growth.
The days on which the last pup in a litter reached such developmental stages were recorded for all litters.
For F3 litters, times of appearance of two additional responses, i.e. righting response and startle response, were recorded.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
A number of deaths occurred in adult rats during the study. Whilst the majority of these deaths occurred in females during pregnancy, there was no indication of a common underlying cause, or any association with treatment.

Dose level (mg/kg/day) M F
0 0 1
50 0 1
500 0 0
1250 0 2
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional differences in body weight between the treated and control groups were seen. In no instance was such a difference consistently present either throughout a generation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Occasional differences in food and water intake between the treated and control groups were seen. In no instance was such a difference consistently present either throughout a generation.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Although a number of organs had different mean weights in treated groups when compared with those of the controls, most of these differences showed no dose relationship or were confined to only one occasion. The main exception was the weight of caecum, both full and empty, which showed a dose related increase both in recorded weight and in weight related to body weight on most occasions.
The mechanism of caecal enlargement is unknown but it has been observed after the administration of a wide range of materials, including other food colourings, and is generally considered to be of no toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At post mortem several lesions were recorded more frequently for the treated animals than for the controls. The finding of red/pink gastro-intestinal contents and yellow caecal contents was consistent in all generations. Other findings, occurring more frequently in the treated groups on one or more (but never all) occasions, were mottled liver, speckled thymus, renal stones and enlarged caecum.
Such findings are generally considered to be of no toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The only treatment-related difference in appearance or behaviour observed was pink coloration of the fur for all dose groups and yellow coloration and softening of the faeces at the two highest doses only.
It is generally considered to be of no toxicological significance.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
The fertility of the treated dams and viability of litters were similar in the treated and control groups in each generation.

Details on results (P0)

Post mortem examination of the dams revealed no differences between treated and control groups.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
A number of deaths occurred in adult rats during the study. Whilst the majority of these deaths occurred in females during pregnancy, there was no indication of a common underlying cause, or any association with treatment.

Adults from F1a
Dose level (mg/kg/day) M F
0 0 1
50 0 1
500 0 0
1250 0 0
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For most generations there were occasional differences in body weight between the treated and control groups. In no instance was such a difference consistently present either throughout a generation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
For most generations there were occasional differences in food and water intake between the treated and control groups. In no instance was such a difference consistently present either throughout a generation.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Although a number of organs had different mean weights in treated groups when compared with those of the controls, most of these differences showed no dose relationship or were confined to only one occasion. The main exception was the weight of caecum, both full and empty, which showed a dose related increase both in recorded weight and in weight related to body weight on most occasions.
The mechanism of caecal enlargement is unknown but it has been observed after the administration of a wide range of materials, including other food colourings, and is generally considered to be of no toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At post mortem several lesions were recorded more frequently for the treated animals than for the controls. The finding of red/pink gastro-intestinal contents and yellow caecal contents was consistent in all generations. Other findings, occurring more frequently in the treated groups on one or more (but never all) occasions, were mottled liver, speckled thymus, renal stones and enlarged caecum.
Such findings are generally considered to be of no toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
During the study, the only treatment-related difference in appearance or behaviour observed was pink coloration of the fur for all dose groups and yellow coloration and softening of the faeces at the two highest doses only.
It is generally considered to be of no toxicological significance.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
The fertility of the treated dams and viability of litters were similar in the treated and control groups in each generation.

Details on results (P1)

The post-mortem examination of the dams revealed no differences between treated and control groups.

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
1 250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
not examined
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For most generations there were occasional differences in body weight between the treated and control groups. In no instance was such a difference consistently present either throughout a generation or during similar phases of different generations.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
For most generations there were occasional differences in food and water intake between the treated and control groups. In no instance was such a difference consistently present either throughout a generation or during similar phases of different generations.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Although a number of organs had different mean weights in treated groups when compared with those of the controls, most of these differences showed no dose relationship or were confined to only one occasion. The main exception was the weight of caecum, both full and empty, which showed a dose-related increase both in recorded weight and in weight related to body weight on most occasions. The effects were much less marked in very young animals and were totally absent in F1b animals autopsied at 25 days old.
The mechanism of caecal enlargement is unknown but it has been observed after the administration of a wide range of materials, including other food colourings, and is generally considered to be of no toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At post mortem several lesions were recorded more frequently for the treated animals than for the controls. The finding of red/pink gastro-intestinal contents and yellow caecal contents was consistent in all generations. Other findings, occurring more frequently in the treated groups on one or more (but never all) occasions, were mottled liver, speckled thymus, renal stones and enlarged caecum.
Such findings are generally considered to be of no toxicological significance.
Histopathological findings:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
During the study, the only treatment-related difference in appearance or behaviour observed was pink coloration of the fur for all dose groups and yellow coloration and softening of the faeces at the two highest doses only.
It is generally considered to be of no toxicological significance.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Viability of litters was similar in the treated and control groups in each generation. Litter development, as gauged by the milestones, showed differences between the treated and control groups in every generation. However, no dose-related differences occurred with F1 pups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1a
Effect level:
1 250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
A number of deaths occurred in adult rats during the study. Whilst the majority of these deaths occurred in females during pregnancy, there was no indication of a common underlying cause, or any association with treatment.

Adults from F1a
Dose level (mg/kg/day) M F
0 0 0
50 0 1
500 1 0
1250 1 1
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For most generations there were occasional differences in body weight between the treated and control groups. In no instance was such a difference consistently present either throughout a generation or during similar phases of different generations.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
For most generations there were occasional differences in food and water intake between the treated and control groups. In no instance was such a difference consistently present either throughout a generation or during similar phases of different generations.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Although a number of organs had different mean weights in treated groups when compared with those of the controls, most of these differences showed no dose relationship or were confined to only one occasion. The main exception was the weight of caecum, both full and empty, which showed a dose-related increase both in recorded weight and in weight related to body weight on most occasions. The effects were much less marked in very young animals. The only other organ-weight differences worthy of note were lower liver weights in females of F2 generation.
The mechanism of caecal enlargement is unknown but it has been observed after the administration of a wide range of materials, including other food colourings, and is generally considered to be of no toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At post mortem several lesions were recorded more frequently for the treated animals than for the controls. The finding of red/pink gastro-intestinal contents and yellow caecal contents was consistent in all generations. Other findings, occurring more frequently in the treated groups on one or more (but never all) occasions, were mottled liver, speckled thymus, renal stones and enlarged caecum.
Such findings are generally considered to be of no toxicological significance.
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
During the study, the only treatment-related difference in appearance or behaviour observed was pink coloration of the fur for all dose groups and yellow coloration and softening of the faeces at the two highest doses only.
It is generally considered to be of no toxicological significance.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
no effects observed

Details on results (F2)

Viability of litters was similar in the treated and control groups in each generation. Litter development, as gauged by the milestones, showed differences between the treated and control groups in every generation. However, the only dose-related differences, consisting of slight delays in the unfurling of ears and eruption of teeth and earlier hair growth, occurred with F2 pups.

The post-mortem examination of the dams and their uterine contents revealed no differences between treated and control groups. Skeletal examinations of the foetuses showed a trend towards slightly earlier ossification in the treated animals in all generations.

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

F3 generation

General health and condition

During the study, the only treatment-related difference in appearance or behaviour observed was pink coloration of the fur for all dose groups and yellow coloration and softening of the faeces at the two highest doses only.

Many rats from the Fla, F1b and F2 litters lost much of their tails during weaning as a result of ringtail.

This disorder did not occur in the F3 litters which were reared in cages with solid-floors rather than the grid-floors

used for earlier generations.

Body weight and food and water intakes

F3 animals showed no treatment-related differences.

Reproductive performance and litter observations

No slight delays in the unfurling of ears and eruption of teeth and earlier hair growth, occurred with F3 generation.

Teratology

Skeletal examinations of F3 foetuses showed a trend towards slightly earlier ossification in the treated animals in all generations.

Post-mortem observations

At post mortem several lesions were recorded more frequently for the treated animals than for the controls. The finding of red/pink gastro-intestinal contents and yellow caecal contents was consistent in all generations. Other findings, occurring more frequently in the treated groups on one or more (but never all) occasions, were mottled liver, speckled thymus, renal stones and enlarged caecum. Overall, such findings are considered of no toxicological significance.

Organ weights

The only other organ-weight differences worthy of note were lower liver weights in F2 and F3 rats and lower adrenal weights in F3 rats. Both of these showed some inconsistencies, the liver difference occurring only in males of the F3 and females of the F2 generations and that for the adrenals being absent in males when the weights were expressed relative to body weights.

Histopathology

Examination of tissues from the high-dose and control F3 animals revealed no differences that could be attributed to treatment. Therefore, tissues from intermediate doses or preceding generations were not examined.

Applicant's summary and conclusion

Conclusions:
Administration of test substance to rats over 3 successive generations at doses up to 1250 mg/kg/day did not produced any toxic effect that could be unequivocally attributed to treatment.
Executive summary:

Method

Test substance was fed to 3 generations of rats, at dietary concentrations to provide 0, 50, 500 or 1250 mg/kg bw/day. In each generation treated groups consisted of 36 rats of each sex while 60 females served as controls. Apart from the F0 generation, which started treatment as weanlings, treatment was continued throughout the study, providing in utero exposure of all offspring. The F0 generation was bred twice, on the first occasion to provide animals for the next generation and for a long-term study, and a second time to provide data on in utero and post-partum development. In each generation approximately one third of the females from each group were killed before parturition to provide data on in utero development. The foetuses from these animals were examined for skeletal abnormalities. Remaining animals were allowed to litter and the offspring were monitored for 21 days after birth for survival and development. All animals were killed and subjected to a post-mortem examination which, for a proportion of each group in each generation, included recording of organ weights.

Results

Deaths of a few adult rats died during the study were not associated with treatment. Fur of the treated animals was coloured pink, faeces and caecal contents of animals from the two highest dose groups were yellow, the faeces also being softer than those of the controls. Such evidences wer attributed to treatment, but were considered of no toxicological relevance.

Treatment had no observed effect on clinical observations, body weight or food and water intake at any stage of the study. Animals fed the two highest doses for prolonged periods had enlarged caeca, but this effect was not seen in weanling animals on the same treatment. Neither the caecal enlargement nor the liver weights seen in the F2 and F3 offspring were considered to be an adverse effect of treatment.

No treatment-related effects were seen in the uterine contents of females at any generation, but the skeletons of treated foetuses showed a slightly more advanced development than those of the controls. Postnatal development of offspring was not affected by treatment at any stage of the study. Tissues from the F3 animals were examined by light microscopy and revealed no treatment-related effects. It was concluded that the no-observed adverse effect level for test substance is 1250 mg/kg bw/day.